ABSTRACT After undergoing positive selection in the cortex, thymocytes migrate into the medulla where central tolerance is enforced by antigen presenting cells (APCs) displaying a diverse array of auto-antigens. Dendritic cells (DCs) and medullary thymic epithelial cells (mTECs) are the two predominant medullary APCs that induce central tolerance. Collectively, mTECs express the majority of the proteome, but any individual self- antigen is expressed by only 1-3 % of these cells. Thymic DCs present self-antigens derived not only from their own proteome, but also those acquired from the blood, from mTECs, and from peripheral tissues. Thus, mTECs and DCs display a mosaic of self-antigens, which thymocytes must navigate to scan for auto-reactivity. This process is critical for self-tolerance, as lost expression of even one self-antigen in the thymus can result in peripheral autoimmunity. In this proposal, we will investigate mechanisms by which the chemokine receptors CCR4 and CCR7 alter thymocyte localization and interactions with distinct APCs to enforce central tolerance. It is well-documented that CCR7 promotes thymocyte medullary localization and thus, negative selection. Over the last funding period, we identified a critical role for CCR4 in these processes as well. Our data suggest a novel model in which CCR4 promotes medullary entry of post-positive selection thymocytes and interactions with DCs, while CCR7 sustains medullary localization of mature thymocytes and promotes interactions mTECs, to induce negative selection and Treg differentiation of distinct TCR repertoires. In Aim 1, we will use a combination of 2-photon microscopy, TCR repertoire sequencing, and TCR retrogenic bone marrow chimeras to test the impact of CCR4 and CCR7 on thymocyte localization, interactions with APCs, and central tolerance. Notably, we have developed a novel 2-photon microscopy approach to quantify the contribution of distinct APCs to negative selection, and will expand this approach to identify APCs required for Treg selection. Our recent data also suggest that both early and late stages of negative selection occur in the medulla, driven by CCR4 and CCR7, respectively. In Aim 2, we will test this novel model, which contrasts with the prevailing view that early and late stages of selection occur in the cortex and medulla, respectively. Over the last funding period, we identified a novel role for CCR7 expression by thymic DCs in regulating Treg selection. In Aim 3, we will use existing and novel genetic mouse models, functional assays, TCR repertoire sequencing, and retrogenic bone marrow chimeras to test the hypothesis that CCR7 expression by thymic DCs promotes their survival and is required for acquisition and display of mTEC-derived self-antigens, thus impacting repertoire selection. Altogether, the proposed experiments will elucidate mechanisms by which CCR4 and CCR7 promote central tolerance and will test the innovative model, suggested by our data, that central tolerance is separated into two stages, first dominated by DC-mediated deletion of less mature thymocytes, and then by mTEC-mediated deletion of mature thymocytes to ensure self- tolerance.